Peripheral clocks are known to modulate circadian patterns of insulin secretion. GLP-1 is an incretin hormone produced by the intestinal L cell that acts as a link between the gut and pancreatic β-cell. Herein, we demonstrate the existence of a diurnal rhythm in GLP-1 secretory responses to an oral glucose load in rats, with increased release immediately preceding the normal feeding period. This profile of GLP-1 release correlated with the pattern in insulin secretion, and both rhythms were completely inverted in animals subjected to a 12-h feeding cycle disruption and abolished in rats maintained under constant light conditions. A daily variation in the insulin response to exogenous GLP-1 was also found. Consistent with these in vivo findings, we demonstrated a circadian pattern in the GLP-1 secretory response to different secretagogues in murine GLUTag L cells, as well as in the mRNA levels of several canonical clock genes. Furthermore, significant changes in the expression of several genes were demonstrated by microarray and knockdown of two of them, thyrotroph embryonic factor and protein tyrosine phosphatase 4a1, resulted in altered GLP-1 secretion. Collectively, these results indicate that an independent peripheral clock in the L cell drives a circadian rhythm in GLP-1 secretory responses.